| Holothuria leucospilota belongs to an animal of Echinodermata Holothuro idea Aspidochirotida Holothuriae,wide distribution,abundant resources,mainly distributed in China's southern Fujian,Taiwan,Guangdong,Guangxi,Hainan Island and Paracel Islands.In order to provide theoretical information for further exploitation and comprehensive utilization of hydrolysis animal protein,the optimum hydrolysis conditions by applying bionic enzymatic hydrolysis of Holothuria leucospilota were investigated;To find the active constituents,and provide reference for its resource-oriented utilization,the chemical compounds in dry Holothuria leucospilota was extracted,separated,purified and identified;And the purification process and characteristics of polysaccharide were preliminarily studied.The whole specimen was air-dried,powdered and researched the optimal process of biomimetic enzymatic hydrolysis of holthuria leucospilota protein.In the optimum hydrolysis conditions,the extract was filtered and evaporated at reduced pressure to obtain a crude residue,which was then successively partitioned between ethyl acetate and water,and between n-butanol and water,to obtain EtOAc-,n-butanol and water-soluble fractions,respectively;The compounds were isolated by repeated column chromatography(CC)on silica gel,Sephadex LH-20,and recrystallization,mainly for ethyl acetate and n-butanol was separated and purified;the structures of these compounds were elucidated by means of the physico-chemical properties and spectroscopic methods.The compounds from Holothuria leucospilota were identified as cholesterol(HS-1),(R)-2,3-dihydroxypropyl stearate(HS-2),cyclo(Leu-Leu)(HS-3),cyclo(Ile-Leu)(HS-4),cyclo(Phe-Leu)(HS-5),cyclo(Pro-Tyr)(HS-6),N-(sec-butyl)-2-phenylacetamide(HS-7),cerebroside HPC-2(HS-8),Botryolide B(HS-9),4-hydroxybenzoic acid(HS-10),uracil(HS-13),thymine(HS-14),2'-deoxyuridine(HS-15),thymidine(HS-16);among which compounds(HS-1)(HS-13)(HS-14)(HS-15)(HS-16),were isolated from Holothuria leucospilota for the first time.The crude polysaccharides from Holothuria leucospilota were obtained by alcohol precipitation after extraction,and the optimum decolorization and deproteinization of crude polysaccharides were studied.The decolorization rate and retention rate of comprehensive consideration,hydrogen peroxide bleaching method was 12.76 higher than activated carbon,the decolorization of hydrogen peroxide is better;Compared with the Sevage method to remove protein,the average yield of polysaccharides was higher by 30.60%.,and has the advantages of simple operation,using acetic acid salt liquid precipitation method is better.And preliminary study on sea cucumber polysaccharide structure characteristics,found holothurian polysaccharide has galactosamine C4 sulfate,mainly composed of glucuronic acid,amino galactose and fucose,holothurian polysaccharide sulfate base,glucuronic acid,amino galactose,fucose content were 29.16%,15.63%,15.03%,10.93%.With the evaluation of the total score of protein content and dry paste yield of holothuria leucospilota as an index,conducting the orthogonal experiment for getting optimum biomimetic enzymatic parameters.The optimum technological conditions for the enzymolysis of holthuria leucospilota were as follows:Under these conditions of material than liquid was 1:8,the reaction temperature was 40?,the pH value of pepsin was 1.5,the pH of trypsin was 8.5,while the dosage of pepsin was 1.0%of the weight of dried holthuria leucospilota powder,the time of enzymolysis was 0.5 h,the dosage of trypsin was 1.5%and the enzymolysis time was 3.5 h.The optimum conditions for enzymatic hydrolysis can reach the maximum total score of protein content and dry paste yield of holothuria leucospilota,and it is reasonable,feasible and efficient. |
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